1. Field of the Invention
The field of the invention is fluorescently-labeled proteins which specifically bind certain phospholipids.
2. Background
The annexins are a family of proteins that specifically bind anionic phospholipids, including phosphatidylserine, in a calcium-dependent manner (Blackwood, R. A. and Ernst, J. D. (1990)Biochem. J. 266, 195-200; Seaton, B. A. (1995) Annexins, R. G. Landes, Austin, Tex.; Cell Mol Life Sci, June 1997;53(6), entire issue). While all annexins bind phosphatidylserine and calcium, they vary in their affinity for phosphatidylserine: for example, at saturating concentrations of calcium (i.e., =1.0 mM), annexin V exhibits a 2- to 160-fold higher affinity for phosphatidylserine compared to other members of the annexin family (Tait, J. F., et al. (1988) Biochemistry 27, 6268-6276; Ernst, J. D., Mall, A. and Chew, G. (1994) Biochem Biophys Res Com 200, 867-876). Annexin binding specificities have been exploited for biological targeting (Tait et al., 1995, J. Biol. Chem. 270, 21594-21599; Oshawa et al, 1996, J. Neurochem. 67, 89-97; Okabayashi et al., 1996, Gene 177, 69-76).
Apoptosis, or programmed cell death, is a universal process that is important in development of multicellular organisms, regulation of the immune system, and clearance of abnormal (including neoplastic and virus-infected) cells (Thompson, C. B. (1995) Science 267, 1456-62). Among the early manifestations of apoptosis in all cell types studied to date is loss of the asymmetric distribution of plasma membrane phospholipids, which results in exposure of anionic phospholipids (including phosphatidylserine) on the extracellular leaflet of the plasma membrane. This exposure of phosphatidylserine, and thus apoptosis, can be detected by various methods, including binding of labeled atinexins (Koopman, G., et al. (1994) Blood 84, 1415-20; Martin, S. J., et al. (1995) J Exp Med 182, 1545-56; Broaddus, V. C., et al. (1996) J Clin Invest 98, 2050-2059; Zhang G, et al., 1997, Biotechniques, Sep;23(3):525-531. Recently, annexin binding specificity has been correlated with other cellular pathology, e.g. King K. B. (1997) J Cell Biochem 65(2), 131-144. Most studies to date have used FITC-annexin V and flow cytometry to identify and enumerate apoptotic cells. Labeling annexin V with FITC requires multiple manipulations of the protein and results in a heterogeneous mixture of labeled protein molecules which vary in the number and position of bound FITC molecules. Moreover, the amino acid residue of annexin V that is most readily available for labeling by FITC is on or near the phospholipid-binding surface, which results in quenching of FITC-annexin V fluorescence by 40-50% upon binding phospholipid membranes (Tait, 1988; Ernst, 1994; supra).
In an effort to circumvent these limitations of FITC-annexins, the present inventor sought to prepare annexins that were labeled homogeneously and that did not change fluorescence properties upon binding membrane phospholipids. Described herein are the preparation and characterization of endogenously fluorescent phosphatidylserine-binding proteins containing Aequorea Victoria green fluorescent proteins (GFPs) fused to annexins. It is shown that these reagents offer highly sensitive detection of apoptotic cells by flow cytometry or fluorescent microscopy, and offer several advantages to chemically modified annexins.
The invention provides methods and compositions relating to Aequorea Victoria GFP-annexin fusion proteins; particularly, recombinant polypeptides comprising a bifunctional green fluorescent proteinxe2x80x94annexin fusion protein providing an equivalent or enhanced measured fluorescent property of the green fluorescent protein and an equivalent or enhanced measured binding specificity of the annexin. In a particular embodiment, the fusion protein comprises a full-length N-terminal GFP fused to a full-length annexin V through a linker comprising an alanine, wherein the fused GFP and annexin moieties provide greater or equal fluorescent intensity and anionic phospholipid binding affinity, respectively, than do the corresponding unfused GFP and annexin proteins.
The invention also provides host cells expressing the subject proteins, including bacteria expressing the subject proteins in soluble form, and methods of using such cells to make the fusion proteins. Uses of the subject fusion proteins include selective cellular and biochemical labeling, particularly anionic species, such as anionic phospholipids. In a particular embodiment, the fusion proteins are used to selectively label apoptotic, dead and/or injured cells.
The subject bifunctional GFP-annexin fusion proteins combine the inherent intense fluorescent properties of green fluorescent proteins with the binding specificity of annexins. The GFPs derive from the jellyfish Aequorea victoria; see e.g. U.S. Pat. No. 5,491,084 for definition, and include variants offering a variety of different excitation and emission wavelengths; see e.g. Heim and Tsien, 1996, Current Biology 6, 178-182. The GFP moiety of the fusion proteins provide an equivalent or enhanced measured qualitative and/or quantitative fluorescent property compared with the corresponding unfused GFP protein. Preferred fluorescent properties are emission and/or excitation peaks, preferably an maximum fluorescent emission peak in unchanged or detectably optimized wavelength and/or undiminshed or enhanced in magnitude or total intensity.
The subject annexins may be derived from a variety of eukaryotic sources, see e.g. Cell Mol Life Sci, June 1997;53(6), entire issue, esp. Liemann S, Huber R, at 516-521 and Morgan R O, at 508-515, and any of the at least thirteen distinct annexin types may be used. The annexin moiety of the fusion proteins provide an equivalent or enhanced measured qualitative and/or quantitative binding specificity compared with the corresponding unfused annexin protein. Preferred binding specificities have equivalent or enhanced affinity for particular anionic cellular components, particularly phospholipids, such as phosphotidylserine.
The GFP and annexin moieties may be separated by a linker peptide, typically from about 1 to 50 residues, which facilitates or at least does not interfere with the requisite bifunctionality of the fusion proteins. The linker may enhance the conformational opportunities of the GTP and annexin moieties and/or provide a third functionality to the fusion protein, e.g. epitopes, post-translational processing sites, etc. Exemplary linkers include alanine or polyalanine, glycine or polyglycine, epitope tags such as FLAG, processing sites such as phosphorylation, ubiquitination or protease recognition/cleavage sites, etc.
Exemplary bifunctional fusion proteins are shown in Table I.
The invention provides recombinant nucleic acids encoding the subject fusion proteins. Typically, natural isolated nucleic acids encoding the GFP and annexin moieties are spliced into expression constructs using conventional methodologies, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) and references cited herein. Alternatively, the amino acid sequences of the subject peptides are used to back-translate peptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166). In either instance, the constructs are designed for expression in any conventional system, such as bacterial, insect, plant and mammalian expression systems. The proteins are preferably secreted and/or expressed in soluble form; preferably most of the protein secreted by or retained within the host cell is in soluble form. Preferred soluble expression avoids denaturation/renaturation and permits single step affinity purification of  greater than 90%, preferably  greater than 95%, preferably in a yield of at least 10, more preferably at least 25 mg/L. In a particular embodiment, the temperature of the expressing host is reduced at least 5, preferably at least 10, more preferably at least 15xc2x0 C. below physiological or environmental temperature for the host (e.g. below 37xc2x0 C. for E. coli or human cells).
Uses of the subject fusion proteins include selective cellular and biochemical labeling, particularly anionic species, such as anionic phospholipids. The subject proteins may be exposed to the targeted cellular or biochemical component in any convenient way, e.g. direct exogenous addition, indirectly by introduction into a cell and expression of a fusion protein encoding nucleic acid, etc., In a particular embodiment, the fusion proteins are used to selectively label apoptotic, dead and/or injured cells.